Cytoplasmic serine/threonine-specific kinases, such as raf and protein kinase C (PKC), play an integrative role in the transduction of signals from the cell membrane to the nucleus. raf and PKC family proteins have a similar structural organization, suggesting analogous domain function and mode of activation. Expression of a chimeric gene consisting of a PKC regulatory domain fused to the raf kinase domain showed that the PKC regulatory domain can regulate the raf kinase in a ligand-dependent fashion. To dissect raf-specific signaling pathways we designed raf inhibition experiments employing monoclonal antibodies as well as expression of antisense RNA. A monoclonal antibody, termed URP26K, generated against the v-raf kinase domain preferentially recognizes the underphosphorylated, inactive form of raf-1. In in vitro assays this antibody blocks PKC-dependent phosphorylation of the raf kinase domain as well as the activation of raf-1. Phosphopeptide mapping revealed that URP26K masks one of two phosphorylation sites specifically induced by TPA treatment in vivo. Expression of raf antisense RNA interferes with proliferation and can revert the morphology of v-raf transformed fibroblasts with a high frequency. Co-expression of a Raf-1 protein, whose kinase activity has been destroyed by a point mutation, proved even more efficient than antisense RNA, suggesting that raf functions necessary for proliferation involve interaction with a rate-limiting ligand or substrate.